Method of preparing polypeptides in cell-free system and device for its realization

ABSTRACT

The present invention provides a method for synthesis polypeptides in a cell-free system by which products of synthesis are branched in a low molecular weight fraction and a fraction which contains high molecular weight components with the target polypeptide, the main part of the low molecular weight fraction is removed via at least one part of the second porous barrier, the ratio of the volume of the fractions of feed solution and expendable components to the volume of the fraction containing the target polypeptide is chosen, modes of supply of the feed solution and expendable components of the fraction are realized.

FIELD OF INVENTION

The present invention concerns to molecular biology and biotechnology,namely to the methods and devices for synthesis of polypeptides incell-free translation system.

BACKGROUND OF THE INVENTION

Several methods of polypeptide synthesis in cell-free translation systemare known. For elimination of restrictions connected with a lower outputof target polypeptides and short-term operation of cell-free translationsystems a method was suggested which is widely used now (Spirin et al.,1988). This method is based on the principle of continuous removal froma reaction mixture of reaction products and continuous restoration ofthe initial concentration of low molecular weight components duringsynthesis. This method underlies several inventions connected with itsimprovement for increasing the synthesized product output (Alkahov etal., 1991; Baranov et al., 1993; Alakhov et al., 1995).

By the input of feeding solutions and removal of products of synthesisthe known methods can be divided as follows: (a) methods in whichdialysis is used to add feed solution components to the reaction mixtureand to remove low molecular weight components from the reaction mixturethrough the dialysis membrane or to simultaneously remove low and highmolecular weight components from the reaction mixture; (b) methods inwhich continuous ultrafiltration is used for a simultaneous removing oflow and high molecular weight components of products through themembrane and a simultaneous input of feeding solutions directly into thereaction mixture volume or through the membrane; c) methods in whichperiodic input of a feed solution into the reaction mixture andsubsequent removing of low and high molecular weight components throughthe membrane are used. Input and output of the flows is realized bychanging the direction of liquid flows at the exposure of consecutivecreation of pulses of positive or negative pressure.

The method (Mozayeny, 1995) is known in which the removal of productswith large molecular weight is improved by increasing the area of aultrafiltration membrane in relation to the reaction mixture volume. Oneof the main disadvantages of the given invention is that during removalof high molecular weight components through the large area of themembrane with the pore size of 70 kD to 100 kD, together with the finalproduct useful working components of molecular weight up to 100 kD arelost. This is a limiting factor for the operating time of the cell-freesystem. The larger is the membrane area, the greater is the amount ofhigh molecular components of the cell-free system washed-off from thereactor at a high flow rate. Another disadvantage is the necessity touse an external loop for creation of a tangential flow of the reactionmixture along the membrane surface. During passage of the reactionmixture via liquid communications three factors influence the work ofthe cell-free systems: (1) when the reaction mixture passes via the loopthe feed solution is not added in the part of external volume of thereaction mixture, (2) low weight products which inhibit the cell-freesystem are not removed from the external volume, (3) the liquidcommunications and pumps are not thermostable and the reaction mixturechanges its temperature depending on the environment. This leads toirreproducibility of results and limits the life time of the cell-freesystem.

The method in which authors offer to apply repeated pulse for input ofthe feeding solution in the reactor and removal of low and highmolecular weight products of synthesis from the reactor via a membraneis known (Fischer et al., 1990). This is realized by changing thedirection of the flow through the membrane. One of the maindisadvantages of the given invention is that low molecular weightcomponents of synthesis which inhibit operation of the system are notremoved from the reactor during a long period. The time during which thefeed solution passes repeatedly via the membrane is equal to the periodwhen a total volume of feed solution passage via the membrane is equalto the complete volume of the reaction mixture. For this purpose Ncycles are formed to create positive and negative pressure. Due to thepressure modulation the inhibiting products come back in the reactortogether with a regular portion of the feed solution. Anotherdisadvantage of this method is that upon formation of N cycles highmolecular weight components of the cell-free system required forprolonged synthesis are intensively washed off the reaction mixture.Thus, repeated returning into the reactor of low molecular weightcomponents inhibits operation of the system and removing from thereactor of high molecular weight components providing effectivesynthesis impose restrictions on operation of system.

The method (Choi. 1997) is known by which synthesis of polypeptides iscarried out with removal of a target product in a dialysis mode ofoperation. For this purpose a membrane divides the reactor in two parts.The reaction mixture is placed on one side of the membrane and the feedsolution on the other side. The reaction mixture is fastly circulatingalong the membrane surface in tangential direction. A disadvantage ofthe method is that due to a large pore size components of the system areremoved together with the target products. Moreover, in spite of thefact that the dialysis process is quite effective because of the largepore size, its extent is not enough for operation of highly efficientcell-free systems.

The method (Alakhov et al., 1991) is known in which amino acids, ATP,GTP in an aqueous buffer are added to reactor during functioning of thesystem and low weight components such as AMP, GDP, Pi formed duringsynthesis and inhibit the system are removed through a membrane. Toprovide a more economical operation of the system, low molecular weightproducts are regenerated and come back into the reactor via themembrane. However from the description and the given figure it is notquite clear how low and high molecular weight components of thesynthesis are removed from the reactor and in what way the buffersolution is regenerated after removal of the polypeptide. Taking intoaccount the description of examples, low and high molecular weightcomponents are removed from the reactor via the ultrafiltrationmembrane. The use of an ultrafiltration membrane is described in anumber of publications (Spirin et al., 1988; Takanori et al., 1991;Spirin, 1992; Erdmann et al., 1994). A disadvantage of this method isthe use of large sizes of the membrane cutoff. In this case highmolecular weight components of systems necessary for synthesis areremoved from the reactor together with target products. The volume oflow molecular weight components is equal to that of the removedcomponents which results in fast closing of the ultrafiltration membranepores.

Methods of adding feed solution to the reaction zone and removing fromit of reaction products for different types of membrane reactors areknown in which the reaction zone is placed between two membranes (Matsonet al., 1988; Wrasidlo et al., 1990; Dziewulski et al., 1992).

The method described in the patent (Alakhov et al., 1995) is theprototype of the method proposed herein. For synthesis of polypeptidesin this invention the reaction mixture is placed between two flatmembranes. The membranes differentiate flows of low molecular weight andhigh molecular weight components and divide the reactor into threezones: zone for input of feed solution, reaction zone, zone of productremoval. The first rather weak flow is formed in the reaction zone. Itensures the reaction mixture movement along the internal part of porousbarriers, through which they molecular weight components (includingsynthesized polypeptides) are removed. The second fast flow is formed inthe zone of feed solution input. It ensures penetration of low molecularcomponents via the membrane in the reaction system. The fast flow of lowmolecular weight components and the slow flow of high molecularcomponents are achieved by creating a tangential flow along the externalsurface of the first porous barrier and dialysis process for adding feedsolution in the zone of synthesis. If high molecular weight componentsare removed from the reactor, the size of cutoff is chosen from 50 to100 kD (which is corroborated by example 7).

The speed of penetration of feed solution components to the reactordetermined to a greater extent by the dialysis process is insufficientfor maintenance of prolonged operation of highly effective cell-freesystems.

Requirements to devices for scientific researches and for synthesis ofpolypeptides in preparative amounts are different. For synthesis ofsmall amounts of polypeptides, from 100 to 200 _(μ)g, it is necessary tohave a simple and cheap reactor which can ensure synthesis during 20-50hours without application of expensive equipment and provides anopportunity to choose hand-operated or controlled speeds or flows.

During synthesis of polypeptides in preparative amounts, the deviceshould control the process: operate speeds of flows inside the reactorand provides an opportunity for a prolonged (more than 50 hours)operation at the expense of active mixing or other action protectingfrom closing of membranes or hollow fibers, provide effective additionof the feed solution and expendable high molecular weight components tothe reaction mixture and effective removal of low molecular weightcomponents from the reactor which inhibit the synthesis.

The device (Mozayeny, 1995) is known which operates in the mode ofcontinuous synthesis of peptides and is controlled by computer. Thesystem includes a complicated and expensive equipment (an automaticsampler etc.).

Devices with one reactor from 1.0 ml. (Spirin et al., 1988) to 100.0 ml(Spirin, 1992) are known. Using the principle of dividing the entirereactor volume in several reactors of smaller volume it is possible toapply identical decisions to devices intended for synthesis ofpolypeptides in laboratory conditions and for prepartive synthesis. Inthis case routine technology of synthesis of polypeptides in smallvolumes of the reaction mixture from 50 _(μ)l up to 1-5 ml can be usedin working with the volume up to 100-200 ml by scaling and increasingthe amount of modules operating in parallel.

Devices for maintenance of synthesis in cells (Puchinger et al., 1980;Gebhard et al., 1997; Hu et al., 1997) using the principle of scalingthe modules are known. In these devices inputs for supplying a feedsolution and outputs for removal of products are connected in parallelfor all N reactors. The known devices are designed for maintenance ofcell growth and cannot be applied for synthesis of polypeptides, as eachof N reaction modules serves to maintain speed, pressure and otherparameters of flows of feed solutions and gases necessary for normalfunctioning of cells.

Known modules for bioreactors on the basis of hollow fibers (Gebhard etal., 1997; Hu et al., 1997) do not take into account the specifity ofworking with cell-free system. Fibers used in the reactors have the samesize of a cutoff and their form reminds a beam placed in a cylinder.Therefore a significant part of the surfaces of hollow fibers contactseach other and reduces the working surface.

The device (Yagihashi et al., 1996) is known whose constructionrepresents two layers of hollow fibers. Each layer consists of gluedhollow fibers placed in parallel. Both layers of hollow fibers have thesame size of a cutoff and a significant part of their surface is incontact.

The device (Pedersen et al., 1994) is known in which separate modulesare single-layer constructions from hollow fibers with the same size ofa cutoff.

However this technical decision has been developed for filtration ofliquids and cannot be used in reactors for cell-free systems withoutessential modification in the design since it is intended for workingwith large volumes of liquid flows.

A large number of designs constructed on the basis of flat membranes isknown. They also have disadvantages being intended basically forfiltration or dialysis.

The device for synthesis of polypeptides in cell-free system is known(Mozayeny, 1995) whose structure includes two flat membranes. Originallythis device (OMEGA TM) was intended for filtration and has a large voidvolume in the zones of product selection. The feed solution and highmolecular weight components are added to the reactor through one input.This device is not intended for assembly in a general constructionconsisting of N modules.

The prototype of the proposed device for synthesis of polypeptides isthe device described in the patent (Alakhov et al., 1985). For synthesisof polypeptides in a mode of product removal the device contains twoporous barriers. These barriers can be executed as flat membranes orhollow fibers. The reaction mixture can be placed both from the externaland internal sides of hollow fibers.

A disadvantage of the given device is that it contains porous barrierswith the same size of a cutoff and provides for removal from the reactorof one flow consisting either of low molecular weight (the cutoff sizeof 7.5 kD) or high molecular weight (the cutoff size up to 100 kD)fractions.

SUMMARY OF THE INVENTION

The presence invention provides a method for synthesis polypeptides in acell-free system by which products of synthesis are branched in a lowmolecular weight fraction and a fraction which contains high molecularweight components with the target polypeptide, the main part of the lowmolecular weight fraction is removed via at least one part of the secondporous barrier, the ratio of the volume of the fractions of feedsolution and expendable components to the volume of the fractioncontaining the target polypeptide is chosen, modes of supply of the feedsolution and expendable components of the fractions are realized.

It is further object of the present invention to describe a methods forsynthesis polypeptides in a cell-free system by which the low molecularweight fraction which consists of removed components including lowmolecular weight components of the reaction mixture and low molecularweight components of the synthesis is withdrawn from the reaction volumevia at least one the second porous barrier only, the mode of supply offractions of the feed solution and expendable components is realized.

It is therefor, also, an object of present invention to provide themethod for obtaining polypeptides in which during the synthesis N cyclesare formed, everyone of which consists of at least two steps, at thefirst step low molecular weight components of the feed solution aresupplied to the reactor via the first porous barrier and the lowmolecular weight fraction with products of synthesis and components ofthe reaction mixture is removed via the second porous barrier, at thesecond step the supply and removal channels are switched and lowmolecular weight components of the feed solution are supplied via thesecond porous barrier, the low molecular weight fraction or the highmolecular weight fraction containing products of synthesis andcomponents of the reaction mixture is removed via the first porousbarrier, the mode of supply of of fractions of the feed solution andexpendable high molecular weight components is realized.

It is a further object of the present invention to describe a reactorwhich comprises at least one reactor volume, whose external surfacecontacts the external surface of the first and second porous barriers,the internal surface of the second porous barrier is connected to thezone of the inlet or outlet of low molecular weight flows, the internalside of the first porous barrier is connected to the zone of the inletand outlet of low molecular weight flows and flows containing highmolecular weight components with the target polypeptide.

BREEF DESCRIPTION OF DRAWINGS

The invention is explained by examples of performance with thereferences to the following figures.

FIGS. 1(a-e) shows a scheme of principles of flow distribution fordifferent modes of operations.

FIGS. 2(a-f) shows block diagrams of modules based on the declaredprinciple.

FIG. 3 shows a block diagram of the device for synthesis in a modewithout removal of the target polypeptide.

FIG. 4 shows a block diagram of the device for synthesis in a mode ofremoval of the target polypeptide.

FIG. 5 shows a block diagram of the device for synthesis in a mode ofremoval of the target polypeptide with a three porous barriers inmodule.

FIG. 6 shows a block diagram of the device for synthesis in a mode withperiodic removal of the target polypeptide.

FIG. 7 shows a histogram reflecting the amount of GFP in the fractionsin accordance with the description in Example 1.

FIG. 8 shows the dependence of the change in the concentration ofsynthesized GFP on the duration of synthesis in a control aliquotaccording to the data of Example 1.

FIG. 9 is a photo of SDS electrophoresis according to the data ofExample 1.

FIG. 10 shows a diagram of dependence of the change in the concentrationof synthesized GFP on the duration of synthesis in a control aliquotaccording to the data of Example 2.

FIG. 11 is a photo of SDS electrophoresis according to the data ofExample 2.

THE LIST OF ABBREVIATIONS

F1 is the feed solution flow.

F10-F20 are flows of low molecular weight components of the reactionmixtures.

Ps are synthesized target polypeptides.

Pc are high molecular weight components of the system.

Rm is the reaction mixture.

Positions 100 are constructive elements of the reactor.

Positions 200 are liquid communications of the device.

Positions 300 are separate elements of the device: values, pumps,compartments for the eluate and compartments for storage of feedsolution.

Position 400 is the controller block

DETAILED DESCRIPTION OF INVENTION

Generally the process can contain all of the following steps:

1. Prepare a reaction mixture on the basis of cell-free prokaryotic oreukaryotic lysates;

2. Prepare a feed solution and expendable components of high molecularweight fraction;

3. Determine the mode of operation of reactor, choose a type of thereactor module with the given types of porous barriers and install adevice for synthesis whose structure includes, at least, one reactormodule;

4. Place the reaction mixture in each of the reaction modules;

5. Depending on the mode of operation determine and set rates of thefeed solution flow and the flow of expendable high molecular weightcomponents through the reaction mixture and speeds of removal of low andhigh molecular weight products;

6. Carry out the synthesis providing the selected modes of operation,during the synthesis continuously or recurrently remove through a porousbarrier the high molecular weight components or leave them in thereactor volume;

7. During the synthesis analyze the yield of the synthesized product,correct the parameters determining rates of feed solution flow and ofthe synthesized product, and supply or not supply expendable highmolecular weight components.

FIGS. 1 (a-e) shows block diagrams explaining the principle of flowbranching in a low molecular weight fraction and a fraction whichcontains high molecular weight components with target polypeptide fordifferent modes of operations.

FIG. 1a shows a block diagram of flow branching inside the module in amode of continuous removal of the target polypeptide. The reactionmodule contains case 100, within which three zones are formed using thefirst porous barrier 110 and second porous barrier 120. The reactionmixture Rm is supplied to reaction zone 130 before the beginning of thesynthesis. The flow of feed solution F1 passes through input 151 inreaction zone 130 and displace high and low molecular weight componentsof the synthesis from the reaction mixture through the first and secondporous barriers. The basic part of low molecular weight fraction passesthrough the second porous barrier 120 with a small size of a cutoff,forming flow F20 removed from the reactor volume through output 142.

Simultaneously high molecular weight components of synthesis Ps,components of cell-free system Pc and part of lower molecular weightcomponents F10 which form the total flow PC+ Ps+ F10 are displaced fromthe reaction mixture through barrier 110 with a large size of a cutoffand through output 162. The ratio between the total volume of feedsolution supplied to the reactor and the volume of the total flow Pc+Ps+ F10 is chosen from 2 to 100. It is preferable that this ratio isfrom 10 to 50. A decrease in the rate of flow through the first barrier110 diminishes formation of a layer of high molecular weight componentsabove the surface of porous barrier 110 and diminishes the process ofclosing of membrane pores by high and low molecular weight components.The concentration of synthesized polypeptides Ps in the reaction mixtureincreases, thus promoting the next step of its clearing. The size of thecutoff of the first barrier 110 is chosen depending on the molecularweight of the synthesized polypeptide and its spatial organization.

FIG. 1b shows a block diagram of flow branching of low and highmolecular weight fractions inside the module in a mode of periodicremoval of the target polypeptide. The reaction module contains case100, inside which three zones are formed using the first porous barrier110 and second porous barrier 120. Before the beginning of synthesisreaction mixture Rm is supplied inside reaction volume 130 through input151. During the synthesis N cycles of flow input/removal through porousbarriers 110 and 120 are formed. Each cycle consists of two steps.During the first step via input 161 the flow of feed solution F1penetrates through the first barrier 110 inside inside reaction volume130. The feed solution displaces low molecular weight components ofreaction mixture and low molecular weight products of synthesis whichform flow F20 from reaction mixture through porous barrier 120 andoutput 142. During the second step the direction of flow input/removalthrough porous barriers 110, 120 is changed. Through input 141 andsecond barrier 120 the flow of feed solution F2 penetrates inside zone130. The pressure of feed solution forms a flow of low and highmolecular weight components of the reaction mixture which passes throughporous barrier 110 and output 162, and flow Pc+ Ps+ F10 is formed. Upontermination of the second step, the cycles comes to its end, the nextcycle begins, or synthesis is terminated. The ratio between the totalvolume of the feed solution, supplied to the reactor during N cycles, tothe volume of total flow Pc+ Ps+ F10 is selected from 2 to 100. It ispreferable that this ratio varies from 10 to 50.

A decrease in the rate of flow through the first barrier 110 diminishesformation of a layer of high molecular weight components above thesurface of porous barrier 110 and diminishes the process of closing ofmembrane pores by high and low molecular weight components. Theconcentration of synthesized polypeptides Ps in the reaction mixtureincreases, thus promoting the next step of its clearing. The size of thecutoff of the first barrier 110 is chosen depending on the molecularweight of the synthesized polypeptide and its spatial organization. Thefirst and second porous barriers are cleaned by changing directions offlows through porous barriers. Duration of a cycle and the temporaryratios between duration of the first and second steps are maintainedconstant during all synthesis, or are changed depending on conditions ofthe synthesis. If the synthesis goes actively and the concentration ofpolypeptides in the reaction mixture quickly grows, to prevent closingof the porous membrane the duration of each cycle is reduced whichautomatically results in more frequent clearing of the pores.

FIG. 1c shows a scheme of flow directions in the mode of operationwithout product selection. During the synthesis all high molecularweight components remain inside the reaction mixture. The feed solutionflow F1 enters through the first porous barrier 110, which has pore sizeup to 1000 kD. The flow of low molecular weight pounds F10, whichinhibiting the cell-free system, is removed via the second porousbarrier 120. The first porous barrier is an allocator flow F1 andprovides uniform input of the feed solution to all points of thereaction mixture.

FIG. 1d shows a scheme directions of flows in the mode of operationwithout product selection for reactor module which have at least one thefirst porous barrier and two parts of second porous barrier. During thesynthesis feed solution flow F1 and/or part of expenable components withhigh molecular weight penetrate through porous of the first barrier 110,which has pore size up to 1000 kD This porous barrier is an allocatorflow F1 and provides uniform input of the feed solution to all points ofthe reaction mixture. The flow of low molecular weight products F10which inhibiting the cell-free system is removed via the two parts ofsecond ports barrier 121-122, which has pore size up to 30 kD.

FIG. 1e shows a scheme directions of flow in the mode of operation withproduct selection for reactor module which have at least one the firstporous barrier and two parts of second porous barrier. During thesynthesis all high molecular weight components including targetpolypeptides is removed from reaction mixture via the first porousbarrier 110, which has pore size up to 100 kD The feed solution flow F1enters through the one part of the second porous barrier 121, which haspore size up to 30 kD. The porous barrier is an allocator flow F1 andprovides uniform input of the feed solution to all points of thereaction mixture. The flow of low molecular weight products F10 whichinhibit the cell-free system is removed via the another part of secondporous barrier 122, which has pore size up to 30 kD.

For realization of the considered method several constructions of thereactor module are offered. The device can provide synthesis of productsin different modes. The module should allow for several conditions:

1. In each point of the reactor at least two processes should be carriedout simultaneously: (a) input of feed solution, (b) removal of lowmolecular weight components which inhibit the synthesis.

2. Input of feed solution and removal of low molecular weight componentsof products should be carried out in the time during which the synthesisdrops below the admissible magnitude.

3. The device should allow effective clearing of the porous membranes orhollow fibers and contain a minimum of void volumes in the liquidcommunications for input/removal of lower/higher molecular weightcomponents/products of synthesis.

For realization of the first and second conditions, a reactor module inwhich whatever thin layers of the reaction mixture can be formed is mostpreferable. The thickness of a layer is chosen from 0.1 mm up to 10 mmprovided that at the given areas of the first and second porous barriersand chosen sizes of their cutoff the average speed of feed solutioninput in the reactor ensures input of the feed solution components inthe most remote points of the reaction mixture in time during which thefeed solution concentration in the remote points drops to the admissiblelevel, and the concentration of low molecular weight componentsinhibiting the synthesis does not exceed this level.

With devices working in the mode in which the feed solution or/and highmolecular weight components move directly to the reactor through atleast one input it is preferable to use additional mixing in thereactor. Such mixing can be performed by a rotating magnetic stir bar ortransfer of the reaction mixture via the external closed loop or othermethods.

For devices in which a thin layer of the reaction mixture is formed, amodule with a separator between layers of porous barriers is mostpreferable. A different type of separator can be used and may beselected from the group of a single or multiple layer, capillarymaterials, a combination of layers of capillary material and porousparticles, a single or multiple thin layer sheets from organic,synthetic or ceramic material, metals or their compositions and porousparticle between layers. Another type of separators may be ofhollow-fibers. Filters widely used for filtration and made of differenttype of cellulose of synthetic polymeric materials, metals and ceramicsmay be also used as separators. The role of layered capillary materialsis not only to divide two porous barriers, but also to increase the areaon which binding of molecules occurs that increases the speed ofreactions connected with the synthesis (Alberts et al., 1986).

It is more expedient to use immobilized porous particles plated on thesurface of layered capillary structures. The diameter of fibers is takenfrom 0.1 up to 0.001 depending on the diameter of hollow fibers.Particles of porous material from 10 microns to 0.1 mm are layered onthe surface of fibers. In case hollow fibers are used as the firstand/or second porous barriers, the fibers of layered materials areaccommodated either along hollow fibers occupying the space betweenthem, or at an angle to the central axes of hollow fibers not exceeding90 degrees (i.e. Across the layers of hollows fibers). The materials ofwhich porous particles can be made include (Choi et al., 1997):polymeric materials (cellulose, gelatin, gollagen), metal compounds andinorganic oxides (aluminum, silica, titanium, zirconium, molybden,vanadium, cobalt) and various zeolites. Porous particles can be used asgranules (Ovodov et al., 1995) on the basis of negatively chargedpolysaccharides and positively charged polymers which formpolyelectrolyte complexes with polysaccharides. Such complexes can beformed, for example, by sodium alginate and poly-L-lysine, sodiumalginate and chitosane, pectin and polyimine, pectin and chitosane. Thematerials of which the porous particles are made may include sorbentsused in chromatography, affinity sorbents can be also used to sreate aporous medium in reaction volume and to isolate the target polypeptidefrom the reaction system after synthesis. Chemical activity andpossibility to inhibit synthesis is restricted for some porous material.

When a cell-free system is immobilized in granules (Ovodov et al., 1995;Alakhov et al., 1995), the most preferable design is the reactor moduleallowing to plate several layers of microganule with immobilizedcell-free system on the surface of the first porous barrier so that theyfill the whole reactor. In this case a rather thin layer ofmicrogranules is formed determined by the height of the reactor, whichallows to supply feed solution to the zone of synthesis and to removethe low and high molecular weight components at the optimal speed.

FIGS. 2a-e shows variants of thin layer formation inside the reactormodule when flat semipermeable membranes and hollow fibers or theircombinations are used as porous barriers.

FIG. 2a shows the case when thin layers of the reaction mixtureareformed between the flat surface of membrane 120 and cylindrical surfacesof hollow fibers that play the role of the first barrier 110 and has theform of at least one layer of parallel hollow fibers. The amount ofhollow fibers depends on the cutoff size of the first and secondbarriers as well as on the diameter of hollow fibers determining theirarea. If the ratio of the cutoff sizes of the first and second barriersis from 3 up to 10, the area of the first barrier is taken from 0.2 to1.0 of that of the second barrier.

FIG. 2b shows the case when the reaction layer is formed between twolayers of hollow fibers functioning as the first barrier 110 and secondbarrier 120. In every layer hollow fibers are placed parallel to eachother. The central axes of hollow fibers placed in different layers areeither parallel or are at an angle from 70 degrees to 110 degrees. Theratio of the amount of hollow fibers in the first layer to the amount ofhollow fibers in the second layer is taken to be from 1 to 0.1 dependingon the ratio of the cutoff sizes. Most preferable is such a design ofthis module when the total volume of reactors is assembled from severalmodules. In this case the modules are located so that layers of hollowfibers from the first and second barriers are alternated, thus allowingto distribute uniformly the flows of low and high molecular weightcomponents via the whole volume of the reactor.

When the synthesis is carried out with cell-free systems whoseefficiency is from 2 to 4 times higher than the known level in 100-200Mg (for example if concentrate reaction mixture is used), it isnecessary to improve the feed solution input to the reactor and decreasethe influence of closing of membrane pores by products of synthesis. Toensure uniform distribution of the feed solution over the entire volumeof the reaction mixture and to lower the closing of the cutoff, a modulewith three porous barriers is used. In this case a layer of hollowfibers parallel to each other is used as the first porous barrier. Thislayer is placed in the middle part of the module between two porousbarriers, which function as the second porous barrier with a small sizeof the cutoff. In this variant the area of the second porous barrier isdoubled, that positively influences the removal of low molecular weightproducts inhibiting the synthesis. This permits to lower the pressureupon the formation of the flow or removed low molecular weight products.

FIG. 2c shows a reactor module in which the reaction layer is placedbetween two flat membranes with a layer of hollow fibers between thelatter. This design of the module is more preferable when one reactorunit is formed from several modules. In this case zones for removal oflow molecular weight components are joined together and the number andlength of liquid communications is reduced.

FIG. 2d shows a design of the module from three layers of hollow fibers.Hollow fibers in each layer are placed parallel to each other. Themiddle layer is placed in such a manner that the central axes of itshollow fibers are at an angle from 70 up to 110 degrees relative to thecentral axes of the other layers or are parallel. It is preferable touse perpendicular allocation of the axes. In this case the area ofcontacting zones between the surfaces of the first, second and thirdlayers of hollow fibers is reduced if no grids or porous layeredmaterials are placed between the layers. The ratio of the amount ofhollow fibers in the first and third layers to the amount of hollowfibers in the second layer is accepted from 1 up to 0.1 depending on theratio of the cutoff sizes of the hollow fibers.

In the above versions of the reactor modules the shape of sheetmembranes and sheets consisting of one-layer hollow fibres can be eithersquare or round. FIG. 2e and FIG. 2f show reactor modules having theform of cylinders in which the reaction volume has the shape of (i) ahelix or (ii) a cylinder.

A difference from the known designs is the formation of a two-layerconstruction with different cutoff sizes and diameters of hollow fibersused. FIG. 2e represents a module in which the central element made of ahollow fiber plays the role of the first porous barrier with a cutoff upto 100 kD. It is coated by fibrous material to prevent direct contact ofthe first and second porous barriers. Then hollow fibers of the secondporous barrier are placed around the central element. The amount ofhollow fibers in the second porous barrier and the area of their surfaceshould exceed those of the first barrier by no less than 5-10 times.Then a beam of hollow fibers is placed in a cover which has an input andoutput for the reaction mixture and its end faces are glued as describedby Yagihashi et al. (1996), forming an output for one porous barrierfrom one side of the cylindrical cover and an output for the otherporous barrier from the opposite side. FIG. 2f shows a two-layer designin which porous or layered material is placed between the two layers.The preliminary prepared sheets of their single-layer constructionsplaying the role of the first and second barriers are curled and placedinside the cylindrical cover with input 151 to the reactor. Internaloutputs of each hollow fiber of the first barrier are united in a commonoutput 161 from one part of the cylinder, and internal outputs of eachhollow fiber of the second barrier are united in a common output on theopposite part of the cylinder.

The form of the reaction module is chosen depending on conditionsproviding for the following: washing the reaction zone, accessible inputof the reaction mixture to the reaction zone without formation of airzones, maintenance of minimal volumes in the zones of input/removal offeed solution and low and high molecular weight components of synthesis.Of great importance is the simplicity of assembly and disassembly of themodule for clearing the cutoffs of membranes and hollow fibers. Anassembly construction (when at least one module is used) is formed byinstallation of membranes, hermetic layers, top and bottom covers intothe frame of the module. It is preferable that one porous barrier ispasted to the frame and the other barrier is removable. This provides anopportunity of easy access to the reaction zone and enables to determinedefects of membranes or hollow fibers that can appear during operation.

It is preferable to use mould materials which can perform two functions,i.e. be a support for porous barriers and hermetically seal the layers.Such materials can be silicon hermetic or other synthetic materials withgood adhesion to polymeric materials from which the membrane and hollowfibers are made. The properties of these materials should providerepeated restoration of the form after elimination of squeezing effort.

Porous barriers can be made of different materials. Nevertheless mostpreferable are those which can allow regeneration and purification ofpores after termination of synthesis without disassembly of the reactoror at its disassembly and subsequent assembly. Clearing of many types ofmembranes and hollow fibers are described in detail in technicalcatalogues of firms (Operating Guide, 1997).

Cutoffs of the first porous barrier 110 are chosen from 30 up to 300 kD.For most polypeptides with molecular weight of 20-40 kD it is morepreferable to use cutoffs of 50-100 kD. Cutoffs of the second barrier120 are taken from 1 up to 30 kD depending on the molecular weight ofsynthesized polypeptides and conditions of passage through this barrierof the given flow of low molecular weight components. For synthesis ofpolypeptides with molecular weight of 20-40 kD it is preferable to usecutoffs of barrier 120 from 10 to 12 kD.

FIG. 3-6 show examples of schemes of devices for different modes ofoperation.

The most simple device for synthesis of polypeptides (FIG. 3) uses amode without removal of high molecular weight components from thereaction mixture. It consists of at least one reaction module 100, pump312 and capacities for the feed solution 315 and waste compartment 316for low molecular weight components. The reaction mixture enters thereactor through input 151. By pump 312 the feed solution is suppliedthrough input 161 and first barrier 110 to the reaction mixture. Lowmolecular weight components inhibiting operation of the system, areremoved from the reactor via second porous barrier 120 and output 142and come to waste compartment 316.

In another example when the experimenter chooses a mode of continuousremoval of the product, the flows are distributed as follows. At thefirst step installation of the system for polypeptide synthesis (FIG. 4)is done using reactor 100 which consists of at least one reaction modulewith two porous barriers, pumps 312 and 314, capacities for storage offeed solution 315, waste fraction 316 and high molecular weight (withthe targed polypeptide) fraction 317. The device works in the followingway. The reaction mixture enters the reaction zone of the module vialiquid communication input 150, the feed solution from capacity 315 issupplied to the next input 151 of this module through liquidcommunications 210 and pump 312 and is distributed over the entirevolume of the reactor, thus displacing products of synthesis. Highmolecular weight products of synthesis, including synthesizedpolypeptides, penetrate through the first porous barrier 110 and areremoved from the reactor via pump 314 with a given speed. Due to thepressure created by pump 312 low molecular weight components are removedthrough the second porous barrier 120 and output 142, which is connecteddirectly to waste compartment 316. The size of the cutoff of the firstand second porous barriers as well as the speed of pumps depend onconditions of the synthesis. The design of the module used for this moderequires the presence of at least two porous barriers in the form of twoflat membranes (FIG. 1a) or two layers of hollow fibers (FIGS. 2b,e,f),or a combination when one of barriers is a membrane and the otherconsists of hollow fibers (FIG. 2a).

When conditions of synthesis require a uniform input of the feedsolution in all points of the reaction zone, a device with reactormodules consisting of three porous barriers is used. Such a device isshown in FIG. 5. The reaction mixture enters the reactor through input151. The flow of feed solution 210 enters the reactor from capacity 315via liquid communications 230, input 142 and porous barrier 121. Thetype of a porous barrier depends on conditions of synthesis. It is moreexpedient that the first barrier 110 is made from hollow fibers and twosecond barriers 121 and 122 are formed from flat membranes or hollowfibers. Through porous barrier 121 the feed solution penetrates into thereactor on the thin layer of the reaction mixture due to negativepressure in the second part of porous barrier 122. The negative pressureis created by pump 312 whose input is connected to output 143 connectedwith internal surfaces of the second porous barrier 122. The output ofpump 312 is connected to waste compartment 316 for low molecular weightcomponents. High molecular weight components of products are removedthrough the first barrier 110 which is made from hollow fibers placed inregular intervals over the thin layer of the reaction mixture. Theinternal part of hollow fibers is incorporated in a common output 161,which is connected to the input of pump 314, whose output is connectedto collector 317 for high molecular weight components.

When it is required to carry out preparative synthesis and the reactionmixture volume is from 1 ml to 500 ml, a parallel connection of Nreaction modules is used. The scheme of a device using a parallelconnection of N modules is shown on FIG. 6. This example is based onperiodic removal of the product when high molecular weight components oftarget products are removed partially at the end of each cycle withsimultaneous automated clearing of pores of the first and second porousbarriers. Reactor 100 is filled with the reaction mixture through input151. In the initial condition valves V3 and V4 are open whereas valvesV1 and V2 are closed. At the first step of a cycle the feed solutionmoves from capacity 315 through pump 312 and valve V3 simultaneously toall N inputs 161.

Then the feed solution passes through internal apertures of hollowfibers or across the internal surface of the membrane from which thefirst porous barrier 110 is formed. Through pores of the first porousbarrier 110 the feed solution penetrates readily to volume with reactionsystem. Low molecular weight components of the synthesis pass throughthe second porous barrier 120 with a small cutoff and are removedthrough N outputs 142 and open valve V4 in waste compartment 316 for thelow molecular weight components. On termination of the first step of acycle and beginning of the second step valves V3 and V4 are closed andvalves V1 and V2 open. Pump 313 creates negative pressure in the channelconnected to valve V2. Under negative pressure, high molecular weightcomponents of synthesis including target polypeptide leave the reactorthrough the first barrier 110 and come to fraction collector 317 viaoutput 161, valve V2 and pump 313. At the same time the feed solutioncomes to the reactor from capacity 315, input 141 and second barrier 120under the action of negative pressure created by pump 313. The flow ofthe feed solution via the second porous barrier 120 at the second stepof the cycle has a different direction than that low molecular weightcomponents formed at the first step of cycles. Therefore pores of thesecond barrier 120 which could be closed at the first step of a cycleopen during the second step of the cycle and hydraulic resistance ofsecond barrier 120 is restored. After termination of the second step ofa cycle its first step is formed. The flow of high molecular weightcomponents via the first porous barrier 110 is stopped. At the firststep of a cycle the flow via first porous barriers changes its directionand pores of the first barrier 110 which could be closed at the secondstep of a cycle by high molecular weight components open and hydraulicresistance of the first barrier 110 is restored. A cycle terminates withthe end of the second step, and the control device monitors switching ofvalves thus giving impetus for the formation of the first step of thecycle. The ratio of the feed solution volume entering the reactor andthe volume of the fraction of high molecular weight components removedfrom the reactor is changed by varying temporary ratios of the times ofthe first and second steps of a cycle and changing its total duration.

It is known (Kim et al., 1996) that by raising the concentration oflysate it is possible to increase the yield of synthesized polypeptides.The data reported in the cited art relate to a batch type reaction, whenthe products inhibiting the cell-free system are not removed from thereaction mixture. This reduces the yield of the synthesized product. Itis possible to raise the yield of polypeptides by removing low molecularweight components of products from the reactor and supplying highmolecular weight components which lose their activity during synthesis.Experiments have shown that one removal of low and high molecular weightcomponents of products from the reactor it is possible to add in thereactor not only the feed solution but such components of lysate asribosomes fraction, extracts (S30, S100 and others), polymerase (T7, T5,SP6 and others), plasmids, tRNA, enzymes or their combinations.

The proposed construction of a reactor module allows to add the feedsolution and lysate components to the reactor in the following ways: (a)the whole volume of feed solution is supplied via the reactor input; (b)the whole volume of feed solution is supplied via the first barrier; (c)part of the feed solution is supplied via the first barrier and theother part via the reactor input; (d) part of the feed solution issupplied via the first barrier during the first step of a cycle and theother part is supplied via the second barrier during the second step ofthe cycle; (e) the whole volume of lysate components is supplied via thereactor input together with the whole volume of the feed solution or itspart; (f) the biggest components of lysate (ribosomes and others) andpart of the feed solution are supplied via the input of the reactionzone while small components of lysate and part of the feed solution aresupplied in the reaction zone via the pores of at least one barrier. Asan example FIG. 5 shows the case when pump 314 can supply high molecularweight components of lysate from capacity 318 as monitored by thecontroller.

Reactor modules are thermostated from 20° C. to 40° C. (the usual rangeis from 25° C. to 37° C.). It is preferable if the feed solutiontemperature is from +2° C. to +7° C. The pumps are hand-operated ormonitored by controller block 400. This block should provide programmingof modes of operation. The automated systems on the basis of computersdeveloped by Roche Diagnostic Boehringer Mannheim have been shown toyield very good (Simonenko, 1998). The controller block allows to adjustthe duration of cycles and the ratio of two steps in a cycle.

At installation, for example, of pressures gauges it is possible totrace the change of pressure in the liquids circuits by the level ofclosing of the cutoff and to change conditions of the process in duetime.

The proposed method of flow branching provides synthesis of polypeptidein a cell-free prokaryotic and eukaryotic extracts with high speedduring tens of hours with removal of functionality active products. Asan example the synthesis of fibrous GFP is given. The synthesis wascarried out with the help of a device whose block diagram is given inFIG. 4. Ultrafiltration membrane with the cutoff from 50 kD to 10 kDwere used as the first and second porous barriers in the reactor module.The volume of the reaction zone was 400 _(μ)l.

In the given example the method of preparative synthesis of polypeptidesin the conjugated system of transcription/translation is used (Baranovet al., 1989). Most frequently estimation of the efficiency of cell-freesystems is made by measuring the amount of radioactive amino acidcontained in the synthesized polypeptides (Alakhov et al., 1991). As analternative method, specific properties of polypeptides such asfluorescence (Kolb et al., 1996; Cramer et al., 1996) are employed.

The S30 extract from E. coli is prepared by a modified method (Zubay,1973) as follows. E. coli A19 cells are grown to optical density of theculture—0.8 at the wavelength of 582 nm. The cells are collected bycentrifugation. The obtained biomass is washed twice being resuspendedin buffer A: 10 mM Tris-Ac pH 8.2, 14 mM Mg(OAc)₂, 60 mM KCl, 1 mMdithiothreitol by centrifugation for 30 min at 10,000 g. The washedbiomass is resuspended in buffer A with the ratio 4 volumes of thebuffer to 1 volume of cells. The obtained suspension is destroyed by theFrench-press at the pressure drop of 1600 bars. Destruction is carriedout at +4° C. Commercial protease inhibitors in concentrationsrecommended by their manufacturers are added to the obtained cellextract, and the mixture is centrifuged for 30 min at 30,000 g.Eliminating agitation, ⅔ of supernatant volume is collected. The volumecollected is centrifuged once again for 30 min at 30,000 g. Eliminatingagitation, ⅔ of the supernatant volume is collected. All procedures arecarried out at +4° C. Buffer B containing 750 mM Tris-Ac pH 8.2, 21 mM(OAc)₂, 7.5 mM dithiothreitol, 6 mM ATP, 500 mM acetylphosphate, 500_(μ)M of each of 20 amino acids. The obtained solution is incubated at37° C. during 80 min. After termination of incubation the extract isdialyzed against buffer C: 10 mM Tris-Ac pH 8.2, 14 mM Mg (OAc)₂, 60 mMKOAc, 0.5 mM dithiothreitol during 16 h. The dialysis is carried out at+4° C. in a 500-fold volume of buffer C with two changes. Aftertermination of dialysis the resulting volume is centrifuged for 30 minat 10,000 g, selected in aliquots and frozen in liquid nitrogen forsubsequent storage at −70° C.

The coupled system of transcription/translation is prepared as follows.

1 ml of the reaction mixture contains 200-400 _(μ)l of the S30 extractfrom E. coli, 0.1-0.5 mg of the total tRNA from E. coli 0.01-0.03 of theplasmid superhelical DNA, 2000-3000 U DNA-dependent RNA from polymeraseof bacteriophage T7, 10-50 U of ribonuclease inhibitor of human placentain buffer D: 50-100 mM HEPES-KOH, or 50-100 mM Tris-Ac, or 50-100 mMTES-KOH, or 50-100 mM MOPS-KOH, or 50-100 mM BES-KOH, pH 7.0-7.6. To thesame reaction mixture we add low molecular weight compounds containing10-20 mM Mg (OAc)₂ or MgCl₂, 120-230 mM KOAc or K-L-glutamate, 1.0-2.0mM ATP, 1.0-2.0 mM GTP, 0.8-1.5 mM CTP, 0.8-1.5 mM UTP, 25-40 mMacetylphosphate, 40 _(μ)g/ml leucovorin, 1 mM dithiothreitol, 4%glycerol, 0.02% NaN₃ and 150-250 _(μ)M of each of 19 amino acidsexcepting the one with which control synthesis in an aliquot of a fixedvolume is carried out.

The prepared transcription/translation system is divided in two unequalvolumes. The smaller volume (30-50 _(μ)l) is placed in microtube and theamino acid containing a radioactive label is added to it. Then aliquotsof 5-10 _(μ)l are taken from the microtube to estimate the kinetics ofthe synthesis in a fixed volume. The amount of synthesized polypeptidesis determined by aliquot precipitation on a glass fibrous filter withtrichloracetic acid, with particles irradiated by the isotope beingcounted in a liquid scintillator. Then the lacking amino acid in theconcentration of 150-250 _(μ)M is added to the remaining volume andafter slight and cautious stirring is placed in the reaction cell.Synthesis is carried out at 26 to 37° C. passing the feed solutionthrough the reaction mixture to the reactor module.

The feed solution is prepared as follows.

1 ml of the feed solution contains the following low molecular weightsubstances: 10-20 mM Mg (OAc)₂ or MgCl₂, 120-230 mM KOAc orK-L-glutamate, 1.0-2.0 mM ATP, 1.0-2.0 mM GTP, 0.8-1.5 mM CTP, 0.8-1.5mM UTP, 25-40 mM acetylphosphate, 40 _(μ)g/ml leucovorin, 1 mMdithiothreitol, 4% glycerol, 0.02% NaN₃ and 150-250 _(μ)M of each of 20amino acids in buffer D. Buffer D contains 50-100 mM HEPES-KOH, or50-100 mM TES-KOH, or 50-100 mM MOPS-KOH, or 50-100 mM BES-KOH, pH7.0-7.6. To the same solution we add 200-400 _(μ)l of buffer Ccontaining 10 mM Tris-Ac pH 8.2, 14 mM Mg (OAc)₂, 60 mM KOAc, 0.5 mMdithiothreitol.

EXAMPLE 1

The coupled transcription/translation of green fluorescent protein (GFP)of coelenterate bacterium Aequoria victoria from a DNA templatecontaining a GFP gene, in continuous cell-free from E. coli, withbranched flows.

A plasmid DNA containing a mutant gene of GFP and nucleotide sequencesof a ribosome-binding site and a promoter of the DNA-dependentRNA-polymerase of bacteriophage T7 is used as a template.

The coupled transcription/translation system is prepared as follows.

1 ml of the reaction mixture containing 200-400 _(μ)l of the S30 extractfrom E. coli, 0.1-0.5 ml of the total E. coli tRNA preparation,0.01-0.03 mg of plasmid superhelical DNA, 2000-3000 U of DNA-dependentRNA polymerase of bacteriophage T7, 10-50 U ribonuclease of humanplacenta inhibitor in buffer E (100 mM HEPES-KOH, pH 7.6). To the samereaction mixture we add low molecular weight substances containing 12 mMMg(OAc)₂, 220 mM KOAc, 1.2 mM ATP, 1.0 mM GTP, 0.8 mM CTP, 0.8 mM UTP,30 mM acetylphosphate, 40 _(μ)g/ml of leucovorin, 1 m dithiothreitol, 4%glycerol, 0.02% NaN₃ and 160 _(μ)M of each of 19 amino acids exceptingleucine.

The prepared transcription/translation system is divided in two unequalvolumes. The smaller volume (30-50 _(μ)l) is placed in a microtube and[¹⁴C]-L-leucine with specific radio-activity 38 mCu/mmol and 100_(μ)M₌concentration is added to it. Then aliquots of 5-10 _(μ)l areselected from the microtube to estimate the kinetics of synthesis in afixed volume. The amount of the synthesized polypeptide is determined byaliquot precipitation on the glass fibrous filter by trichloroaceticacid with subsequent estimation of irradiated particles in a liquidscintillation counter. Leucine in 160 _(μ)M concentration is added tothe remainder volume and after sight and cautious mixing the preparationis placed in a reaction cell.

The synthesis is carried out at 26° C. in the reaction cell. The feedsolution is supplied to the reaction cell at a rate equal to its 1.5-2internal volumes per 1 hour. The product is removed at a rate equal to{fraction (1/20)}-{fraction (1/8 )} of the internal volume of the cellper 1 hour. During the entire synthesis the specific fluorescence of theproduct removed through a membrane with the cutoff of 50 kD is recorded.The efficiency of the system is estimated by fluorescence of allassembled volumes. FIG. 7 represents a histogram showing the amount ofGTP in fractions collected during synthesis. The amount of GFP in thefractions is estimated by the calibration curve plotted using the dataof specific fluorescence measurements of the purified GFP preparationversus its concentration. FIG. 8 shows a diagram of dependence of achange in the concentration of synthesized GFP on the time of synthesisin a control aliquot incubated in a constant volume. The concentrationis determined by incorporation of radioactive amino acid in thepolypeptide. In addition the newly synthesized product is controlled byregistration of fluorescence after electrophoresis of all assembledvolumes in polyacrylamide gel. FIG. 9 shows a photo of gelelectrophoresis with aliquots selected from fractions collected duringsynthesis. A purified GFP preparation is used as control. The amount oflayered protein is 0.3 mg.

A portion of 150-200 _(μ)g of a functionally active product is obtainedduring 48 h per 1 ml of the coupled transcription/translation system.

EXAMPLE 2

The coupled transcription/translation of green fluorescent protein (GFP)of coelenterate bacterium Aequoria victoria from a DNA templatecontaining a GFP gene in a continuous cell-free system from E. coli withone flow.

The DNA is obtained and the coupled transcription/translation system isprepared as described in Example 1. The synthesis is carried out using adevice whose block diagram is given in FIG. 3. Hollow fibers of 1 mm indiameter and the cutoff size of 100 kD were used as the first porousbarrier in the reactor module. An ultrafiltration membrane with thecutoff size of 10 kD and the area of 4 cm² were used as the secondporous barrier. The reactor volume was 400 _(μ)l.

The feed solution flow is supplied to the reaction cell at a rate equalto its 1.5-2 internal volumes per 1 hour. The product is not removed.

The efficiency of the system is estimated from the fluorescence of thesolution removed upon termination of synthesis from the reaction cellwith a subsequent determination of the amount of the polypeptidessynthesized. Then it is compared to the efficiency of control synthesisin a fixed volume. FIG. 10 shows a diagram of dependence of a change inthe concentration of synthesized GFP on the duration of synthesis in thecontrol aliquot incubated in a constant volume. The concentration isdetermined by incorporation of radioactive amino acids in thepolypeptide. In addition the newly synthesized product is controlled byrecording fluorescence after electrophoresis of all collected volumes inpolyacrylamide gel. FIG. 11 represents a photo of gel electrophoresiswith aliquots selected from the coupled transcription/translation systemin fixed volume and from the reaction cell after termination ofsynthesis. A purified preparation of GFP is used as control. The amountof layered protein is 0.3 mg.

During synthesis 60 _(μ)g of a functionally active product are obtainedfor 24 h per 1 ml of the coupled transcription/translation system.

REFERENCES

Spirin A. S. et al., A Continuous Cell-Free Translation System Capableof Producing Polypetides in High Yield. Science 242, 1162-1164 (1988)

Spirin A. S. Cell-Free Protein Synthesis Bioreactor. From Frontiers inBioprocessing II, 31-43, Editors: Todd P. et al. Amer. Chem. Soc. (1992)

Takanori K. et al. A Continuous Cell-Free Protein Synthesis System forCoupled Transcription-Translation. J. Biochem. 110, 166-168 (1991)

Erdmann V. A. et al. The Protein-Bioreactor: Its Potentials for theSynthesis of Proteins in Biotechnology. Medicine and Molecular Biology,First German-Russian Summerschool on In vitro Systems, 64-70, Berlin(1994)

Kim D. et al. A highly efficient cell-free protein synthesis fromEscherichia coli. Eur. J. Biochem. 239, 881-886 (1996)

Flux Recovery-Cleaning, Sanitization, Storage, Depyrogenation, fromUF/MF Operating Guide, A/G Technology Corporation, 16-20 (1997)

Alberts B. et al. Molecular Biology of a Cell. p. 133, New York, London(1983)

Simonenko P. et al., Controlled System for Cell-free Protein Expression:Protein Biosynthesis Reactor, ??? (1998)

Baranov V. I. et al. Gene Expression in a Cell-free System onPreparative Scale. Gene 84: 463-466 (1989)

Kolb V. A. et al., Synthesis and Maturation of Green Fluorescent Proteinin a Cell-free Translation System, Biotech. Lett. 18: 1447-1452 (1996)

Zubay G. In vitro Synthesis of Protein in Microbial Systems, Annu. Rev.Genet. 7: 267 (1973)

Crameria A. et al., Improved Green Protein by Molecular Evolution UsingDNA Shuffling, Nature Biotech. 14: 315-319 (1996)

PATENTS

Alakhov Yu. B. et al, Method of preparing polypeptides in a cell-freetranslation system. U.S. Pat. No. 5,478,730, Dec. 26, 1995, U.S.Cl.—435/68.1.

Choi C. et al. Method producing protein in a cell-free system. U.S. Pat.No. 5,593,856, Jan. 14, 1997, U.S. Cl.—435/68.1

Mozayeni B. R. Apparatus and process for continuous in vitro synthesisof proteins U.S. Pat. No. 5,434,079, Jul. 18, 1995, U.S. Cl.—435/311

Fischer K. H. et al. Verfahren zur Beschleunigung des Stoffaustauschseines kontinuierlichen Bioreaktors and Vorrichtung zur Durchfuhrungdieses Verfahrens. DE Pat. No. 39-4956 A1, Nov. 22, 1990, Int. Cl.—C12 M1 {fraction (1/12 )}

Alakhov Y. B. et al. A method of preparing polypeptides in cell-freetranslation system. SU—1618761 A1, 07.01.91, N1 1991, Int. Cl.—C 12 P21/02

Baranov V. I. et al. A method of preparing polypeptides in cell-freetranslation system. EP Patent 0593757, 15.01.1997, Int. Cl. C12P 21/00

Matson S. L. et al. Method and apparatus for conducting catalyticreactions with simultaneous product separation and recovery. U.S. Pat.No. 4,786,597, Nov. 22, 1988, U.S. Cl. 435/041

Wrasidio W. J. et al. Thin film membrane enzyme reactor and method ofusing same. U.S. Pat. No. 4,956,289, Sep. 11, 1990, U.S. Cl. 435/180

Dziewulski D. et al. Three compartment bioreactor and method of use.U.S. Pat. No. 5,135,853, Aug. 4, 1992, US Cl. 435-04

Hu W. et al. Bioreactor device with application as a bioartificialliver. U.S. Pat. No. 5,605,835, Feb. 25, 1997, U.S. Cl. 435-297,2

Puchinger H. et al. Process for the in -vitro biosynthesis of hormones,especially insulin. U.S. Pat. No. 4,225,671, Sep. 30, 1980, US Cl.435/71

Pedersen S. K. et al. Cartridge of hybrid unitary wafers of hollow fibermembranes and module containing a stack of post-potted cartridges. U.S.Pat. No. 5,366,625, Nov. 22, 1994, U.S. Cl. 210-321.78

Hargitay B. Ultrafiltration and reverse osmosis device comprising pluralcarbon tubes bonded together. U.S. Pat. No. 4,341,631, Jul. 27, 1982,U.S. Cl. 210-323.2

Yagihashi T. et al. Separation module and bundle unit of hollowthread-type porous membrane elements and methods of producing same. U.S.Pat. No. 5,584,997, Dec 17, 1997, U.S. Cl. 210-321.79

Hanai T. Bundle of permselective hollow fibers and a fluid separatorcontaining the same. U.S. Pat. No. 5,198,110, Mar. 30, 1993, U.S. Cl.210-321.79

Gerbhard T. et al. Multi-bioreactor hollow fiber cell propagation systemand method, U.S. Pat. No. 5,656,421, Aug. 12, 1997, U.S. Cl. 435/003

Ovodov S. J. et al. Method for obtaining polypeptides in a cell-freetranslation system. EP Pat. EP 0 485 608 B1, 22. 11.95, Bulletin 95/47,Int. Cl. C12P 21/00.

What is claimed is:
 1. A device for synthesis of polypeptides in acell-free translation system containing at least one reactor moduleincluding a reactor volume having an inlet and an outlet for linkingliquid circuits and including at least two porous barriers, a system forsupply of different fractions of the feed solution and expendable highmolecular weight components, consisting of at least one pump, liquidcircuits, compartments for accumulation and storage of products or feedsolution, wherein the reactor comprises at least one reactor volume,whose external surface contacts the external surface of the first andsecond porous barriers, the internal surface of the second porousbarrier is connected to the zone of the inlet or outlet of low molecularweight flows, the internal side of the first porous barrier is connectedto the zone of the inlet and outlet of low molecular weight flows andflows containing high molecular weight components with the targetpolypeptide.
 2. The device according to claim 1 wherein each reactormodule has a reactor volume from 50 μl to 10 ml and the thickness of thereactor layer from 30 μm to 5 mm.
 3. The device according to claim 1wherein each reactor volume has the shape of either (i) a rectangularsheet, or a square sheet, or a round sheet, or (ii) a sheet foldedeither in a helix or cylinder.
 4. The device according to claim 1wherein the ratio of the area of the surface of the second porousbarrier to the area of the surface of the first porous barrier is from 1to
 100. 5. The device according to claim 1 wherein each reactor volumecontains either (i) the reaction mixture, (ii) the reaction mixture witha magnetic stirrer, or (iii) the reaction mixture with a porousextender, or (iv) the reaction mixture with porous granules.
 6. Thedevice according to claim 3 wherein each extender is made of porousmaterials with pore sizes from 200 μm to 10 nm, these materials areselected from (i) fibrous structures in the form of bundles or sheets,(ii) a composition of fibrous structures with a coating of porousparticles, (iii) affinity sorbents in the form of granules orcomposition of fibrous structures with a coating of affinity sorbents,and (iv) granules with immobilized components of the cell-free system.7. The device according to claim 1 wherein the above first and secondporous barriers are made either from flat membranes or from hollowfibres, or one barrier is made from a membrane and the other barrier ismade from hollow fibres.